1. Field of the Invention
The present application concerns high-speed mechanisms for automatically identifying and physically selecting multicellular organisms or other large objects with predetermined characteristics from mixed populations and depositing them in discrete locations.
2. Description of Related Art
Intact multicellular organisms, such as nematodes, fruit fly larvae, or zebrafish embryos are frequently used as model systems to help understand the function of human genes that have been implicated to play a role in disease. Human gene homologous have been identified in these model organisms and mutations have been induced specifically in those gene homologous. Such mutations frequently result in an easily observable phenotypic change in the model organism, and it has been shown that certain mutants respond to pharmacological compounds with a measurable mode of action. Mutants of intact organisms are now used as a new class of in vivo drug screens for combinatorial pharmacological compound libraries. By using these organisms, one can identify targets for drug intervention without the need to completely understand complex biochemical pathways between the genotype and the phenotype. In addition solid state combinatorial chemical approaches are now being utilized to produce these drug libraries; the end result is that the sample chemicals to be tested are present on solid microspheres usually between 100 and 500 xcexcm in diameter. These solid state techniques greatly speed the preparation of the sample compound library but necessitate a method to accurately select and dispense these microspheres for testing purposes.
The historic approach to modeling diseases in multicellular organisms has been to make morphological or behavioral mutants with substantial phenotypic defects. The intent of such research is to produce a mutant that resembles or models a disease state so that new therapeutics can be screened without using human xe2x80x9cguinea pigs.xe2x80x9d In fact, considering the current prevalence of animal rights activists, the safest approach is to entirely eschew the use of mammals for testing purposes. The goal, then, has been to observe these model disease defects and their interaction with candidate therapeutics objectively and with high sensitivity. Unfortunately, this goal has been not often met. The closest approach to reaching the goal has been to devise xe2x80x9clive-deadxe2x80x9d assays that can be carried out in microwell arrays using optical readout systems. The plan is to dispense individual organisms into microwells, add the candidate therapeutic and optically detect the response. If the candidate therapeutic is present on a microsphere, then the microsphere must also be accurately selected and dispensed.
The exposure of model organism mutants to diverse pharmaceutical compound libraries, even when the mutation has not been linked to a human gene homologue also helps define gene function. The addition of such functional genomic techniques to the repertoire of molecular biology and biochemistry methods is leading to a significant increase in speed in the pharmaceutical discovery process. Investigators annotate pharmaceutical drug libraries for toxicity, non-specific activity, or cell membrane permeability, etc. by observing their behavior in intact organisms. This way, potential new therapeutics that show toxicity or harmful results can be discarded early without wasting valuable resources.
The soil nematode Caenorhabditis elegans, has become a particularly important multicellular organism for these types of tests because its anatomy, development, behavior and genome, is more completely understood than that of any other animal. C. elegans is a small metazoan animal composed of only 959 cells, each generated from a single zygote cell through a completely known cell lineage. This small number of cells nonetheless exhibits a diversity of cell types that typifies more complex animals, including skin, muscle, gut and nerve cells.
The genes of C. elegans are easily accessed through powerful classical and molecular genetic tools. The sequencing of the C. elegans genome is also more advanced than that of any other animal and is a model for the Human Genome Project. Although most human disease genes that have been identified and cloned based on chromosomal position have no known function, the vast majority of these as well as most other human genes have C. elegans homologs. These homologs can be rapidly analyzed using the above-mentioned approach to elucidate the functional biology of the homologous human gene.
A striking conclusion from studies of C. elegans is that the cellular and molecular mechanisms that operate in this nematode are strikingly similar to those that operate in more complex animals, including man. These similarities are so great that homologous human genes can function in nematodes and nematode genes can function in mammalian cells. Researchers are therefore using this nematode for numerous types of experiments related to the development of pharmaceutical agents for use in humans and other higher animals.
Despite the potential power and speed of using multicellular organisms like C. elegans current programs for rapid pharmaceutical drug discovery of not employ high-speed preparation techniques. As an example, with today""s molecular biology techniques, a large laboratory can produce deletion mutations in multicellular organisms at a rate of 20 to 30 per month. To evaluate the effect of a chemical compound library (that frequently may contain 100,000 or more members) on a class of mutated organisms, one must first manipulate and deposit a precise number of organisms in the same development stage into a container, such as the wells of a microtiter plate array. Organisms of different development stage must be excluded since they would convolute the measured response.
Using slow, manual methods, the selection and deposition of organisms of the proper type is a bottleneck for the entire process of pharmaceutical discovery. If the test compounds are present as microspheres, then the accurate selection and dispensing of microspheres adds an additional bottleneck. Furthermore, manual methods rely on pipettes that dispense accurate volumes of fluid and not accurate numbers of organisms. In many studies where reproduction rate is altered by the mutation, it is necessary to begin the study of the effect of a compound from the combinatorial library with an exact, and known number of multicellular organisms in each well. Any selection system based on volume is liable to dispense inaccurate numbers of organisms because precisely uniform suspensions of organisms are impossible to maintain. In the same way if the test compounds are available as microspheres it is extremely difficult to place a controlled number of microspheres in each well. Further, the microsphere population may be mixed so ultimate results require not only precise counting but selection of microspheresxe2x80x94clearly an impossible task for simple pipettes.
Flow cytometers have operational characteristics that make them adaptable to the problems of automating the selection and deposition of multicellular organisms and other large objects such as microspheres. Flow cytometers have been used to count the number of nematodes in a given volume of fluid. Such a device was described by Byerly et al (Byerly, L., R. C. Cassada, and R. L. Russell, xe2x80x9cMachine for Rapidly Counting and Measuring the Size of Small Nematodesxe2x80x9d, Rev. Sci. Instrum. Vol 46, No. 5, May 1975) where the flow cytometer utilized sheath flow to orient the nematodes along the direction of flow so that their length could be measured and organism-by-organism counts could be made by an electrical impedance method similar to that used in a commercial Coulter(copyright) counter. A flow cytometer for working with multicellular organisms is not limited to using an impedance sensor, biut can be a more modern optically sensing flow cytometer.
For example, an optical flow cytometer for analyzing elongate organisms such as plankton with widths of 500 xcexcm and lengths over 1000 xcexcm has been described in a number of published articles such as Peeters, J. C., G. B. Dubelaar, J. Ringelberg, and J. W. Visser, xe2x80x9cOptical Plankton Analyser: a Flow Cytometer for Plankton Analysis, I: Design Considerationsxe2x80x9d Cytometry Sept 10 (5): 522-528 (1989); and Dubelaar, G. B., A. C. Groenwegen, W. Stokdijk, G. J. van den Engh, and J. W. Visser, xe2x80x9cOptical Plankton Analyser: a Flow Cytometer for Plankton Analysis, II: Specificationsxe2x80x9d, Cytometry Sept 10 (5): 529-539 (1989). The size range of the plankton used in these optical flow cytometers is similar to that encountered with nematodes, fruit fly larvae, and zebrafish embryos. In all of these references, the multicellular organisms were merely analyzed but were not selected and deposited. Similarly, analysis of large microspheres with flow cytometers is routine as long as the cross-sectional area of the flow cell is sufficient to accommodate the microsphere.
Selection and deposition of non-multicellular organisms and other small objects with flow cytometers is well known. The method used to select and deposit specific organisms or objects (e.g. microspheres) on command from the flow cytometer consists of a mechanism to switch the direction of the flowing stream of organisms or objects that emerges from the flow cell of the flow cytometer so that analyzed objects can be specifically deposited in a microwell plate or similar container. Switching is performed at a fixed delay time after the flow cytometer has identified a desirable organism. The delay is typically in the time scale of a millisecond to tens of milliseconds. The most common method found in commercial cell sorters is electrostatic diversion of desired objects once they have emerged from an exit port in the flow cell into air. Electrostatic diversion is accomplished by charged plates that operate on a stream of droplets.
However, electrostatic cell sorters are designed specifically for single cells and are not useful for sorting large objects such as nematodes, fruit fly larvae, zebrafish or large microspheres. This is because the flow cell of an electrostatic cell is mechanically vibrated at frequencies of tens of kilohertz to mechanically break the fluid stream into (charged or uncharged) droplets in air that are of the order of 50 xcexcm in diameter. This size droplet is optimal for typical single cells with diameters of 5 xcexcm to 30 xcexcm, but it is much smaller than most multicellular organisms, which are typically of the order of 1 mm in length. The mechanical vibration step and the subsequent breakup of the stream into small droplets is typically lethal to multicellular organisms. The vibration frequency of an electrostatic cell sorter is not variable; therefore, one cannot change the droplet size to accommodate multicellular organisms. Furthermore the entire flow cell always vibrates at this frequency, making it impossible to create single droplets on command.
In the case of large microspheres used in combinatorial chemistry there is no worry that mechanical vibration will damage the microsphere. Nevertheless, electrostatic sorters are unable to effectively select and deposit such large objects. This is a result of the geometry used with the electrostatic deflection plates. At the voltages commonly used static charge results in a deviation of only a few degrees. It is impossible to produce greater deviations by increasing the voltages because arching will occur. Adequate deviation to separate selected from rejected droplets is achieved by allowing the stream to fall a sufficient distance beyond the charged plates. In the case of the typical 50 xcexcm droplet the droplets fall an additional 2.5 cm beyond the deflection plates. If the droplet size is doubled to 100 xcexcm (still insufficient to accommodate a 100 xcexcm combinatorial chemistry microsphere), the larger droplet has greatly increased mass which means that the angle of deviation is smaller; therefore, a longer fall distance is necessary to produce adequate deflection (i.e., the deflection angle is smaller). The net result is that 100 xcexcm droplets require a fall distance of 20 cm. With such a large fall distance tiny instabilities in the flow stream are magnified into appreciable deflections. The microwells of the plates in current use may be on the order of one to a few millimeters in diameter. With a 20 cm fall distance current electrostatic sorters are unable to accurately hit such a small target. The problem becomes even more acute when the droplet size is increased farther to accommodate 400 xcexcm microspheres or multicellular organisms. With a droplet size of one-millimeter (the size necessary to cushion a typical nematode) the fall distance increases to about 125 cm making it totally impossible to deposit droplets in target containers of even several millimeters diameter.
Thus, electrostatic sorters are completely unsuited to multicellular organisms or other large objects. Even if the process does not kill or damage the organism, the deflection geometry makes it impossible to accurately deposit large objects.
The invention features an instrument for selecting and accurately dispensing multicellular organisms and other large objects. The instrument uses hydrodynamic flow conditions in an alignment chamber to align elongate multicellular sample organisms and center organisms or objects in the center of a fluid flow stream after which they pass single file through a sensing zone which is preferentially within the chamber. In the sensing zone the aligned and centered objects are interrogated preferably by a light beam. Optical detectors receive refracted, reflected, fluoresced and scattered light from the interrogated objects and output corresponding electrical signals. A signal processing computer system uses these signals to choose desired analyzed objects. A first fluid switch downstream of the sensing zone and outside of the chamber is responsive to signals developed by the computer system. When the switch is open, the flow stream containing the objects passes the switch and into a collection container. When the switch is closed, a fluid stream from the switch deflects the flow stream containing the analyzed objects and prevents it from reaching the collection container.
In preferred embodiments, the fluid switch can include a switched source of compressed gas having a gas output directed toward a location downstream from the sensing zone and outside of the chamber. The switched source of compressed gas can include a source of compressed gas and an electrically operated valve, such as a solenoid valve, to interrupt a gas stream from the source of compressed gas. The switched source of compressed gas can be operative to interact with the fluid flow stream carrying objects from the sensing zone with sufficient force to convert the carrier fluid into a droplet spray. A sample source can be operative to supply a fluid carrying a sufficiently low concentration of large sample objects that the objects flow substantially one at a time through the sensing zone. The fluid switch can be responsive to a delayed detection signal from the computer system. The fluid switch can be operative to include only predetermined amounts of fluid with the selected sample object. The computer system can be operative to cause the switch to select one object at a time, with each object being accompanied by a predetermined volume of fluid.
An illumination source can be directed toward the sensing zone, with the detector being an optical detector. The computer system can be operative to determine the length of at least one of the selected objects by measuring the time that the at least one of the objects takes to pass between the detector and the illumination source. The detector can be an on-axis detector, located across the sensing zone along an illumination axis of the illumination source. The detector can be an off-axis detector generally perpendicular to an illumination axis of the illumination source. An on-axis detector can be located across the sensing zone along the illumination axis of the illumination source. The illumination source can be a focused low-power laser. The sensing zone can have a width of about 10-40 xcexcm. The sensing zone can have a square cross-section. The output opening of the sample source can be separated from the sensing zone by a total conduit volume of less than 500 microliters. A second fluid switch downstream of the first fluid switch and outside of the chamber can dispense the selected objects into different containers.
In another general aspect, the invention features a multicellular organism or large particle dispensing instrument that includes means for aligning the organisms or objects in a fluid stream in a direction parallel to a flow direction of the fluid stream, means for detecting the presence of the organisms or objects in the fluid stream located downstream from the means for aligning, and means for selectively diverting portions of the fluid, with the means for selectively diverting being located downstream from the means for detecting, being outside of any chamber containing the means for aligning and being responsive to the means for detecting.
In preferred embodiments, the multicellular organism and large object dispensing instrument can further include means for redirecting an output of the means for determining relative to a first container to thereby dispense further ones of the organisms into a second container. The means for selectively diverting can be for including only a predetermined amount of fluid with each of the organisms selected.
In a further general aspect, the invention features a method of dispensing multicellular organisms and large objects that includes centering and orienting the organisms or objects in a longitudinal orientation in a chamber, flowing the organisms in the longitudinal orientation through the center of a sensing zone with a carrier fluid, and detecting the presence of the organisms or objects in the sensing zone. At least some of the carrier fluid is diverted by means for diversion based on the step of detecting ones of the organisms or objects and ones of the organisms or objects remaining in portions of the carrier fluid that were not diverted are collected. The means for diversion are disposed outside of the chamber.
In preferred embodiments, the step of diverting can include a step of converting the carrier fluid into a droplet spray. The step of diverting can take place for a predetermined period of time for each of the detected organisms. The method can also include step of illuminating the sensing zone, with the step of detecting light from the step of illuminating. The step of detecting can employ an on-axis detector and an off-axis detector and combine signals from these detectors. The step of centering can include a step of conveying a sheath fluid past a nozzle. The step of conveying can be performed with a maximum Reynolds number of around one hundred. The method can further include a step of sorting the organisms or objects into a plurality of categories after the step of diverting, with the step of collecting placing the organisms or objects in a plurality of different containers. The method can further include the step of exposing the organisms collected in the step of collecting to a pharmaceutical agent, which may be borne by a large object. The step of dispensing the organisms can include dispensing predetermined numbers of nematodes into each of a number of containers. The step of flowing can introduce reference particles along with the nematodes. The step of dispensing can include dispensing only multicellular organisms having a particular characteristic into a given container.
In another general aspect, the invention features a dispensing instrument that includes a source of organisms or large objects, a sensing zone responsive to presence of organisms or objects, a detector directed toward the sensing zone, and a first switched source of fluid having an output directed toward a location downstream from the detector and having a control input responsive to the detector.
In preferred embodiments, the switched source of fluid can include a source of compressed gas and an electrically operated valve, such as a solenoid valve, to interrupt a gas stream from the source of compressed gas. The switched source of fluid can be operative to interact with a fluid stream from the detector with sufficient force to convert fluid in the detector fluid stream into a droplet spray. The switched source of fluid is not contained within any flow chamber so as not to introduce fluidic instabilities. The switched source of fluid can be responsive to a delayed detection signal from the detector. The dispensing fluid switch can be operative to repeatedly leave predetermined amounts of detector fluid stream fluid undiverted. The dispensing instrument can further include a second switched source of fluid positioned to divert fluid left undiverted by the first switched source of fluid.
In a further general aspect, the invention features a dispensing instrument that includes means for providing a fluid stream carrying objects, the means for providing being located within a flow chamber, means for detecting the presence of the objects in the fluid stream, the means for detecting being located downstream from the means for providing, and first means for selectively directing a gas stream toward the fluid stream to divert portions of the fluid, the means for selectively directing being located downstream from the means for detecting, outside of the chamber, and being responsive to the means for detecting.
In preferred embodiments, a second means can be provided for selectively directing an output of the first means for selectively directing, relative to a first container to thereby dispense portions of the fluid stream into a second container. The means for selectively diverting can be for including only a predetermined amount of fluid with each of the objects selected.
In another general aspect, the invention features a dispensing method that includes feeding objects through the center of a sensing zone with a carrier fluid, detecting the presence of the objects, diverting at least some of the carrier fluid based on the step of detecting, and collecting ones of the objects remaining in portions of the carrier fluid.
In preferred embodiments, the step of diverting can include a step of converting the carrier fluid into a droplet spray. The step of diverting can take place for a predetermined period of time for each of the objects. The step of diverting is physically removed from the step of detecting so as to avoid introducing fluidic instability. The method can further include a step of sorting the objects into a plurality of categories after the step of diverting and the step of collecting can collect the objects in a plurality of different containers. The method can further include the step of exposing the objects collected in the step of collecting to a pharmaceutical agent. The step of dispensing the objects can include dispensing predetermined numbers of the objects into each of a number of containers. The step of feeding can feed reference particles with the objects. The step of dispensing can include dispensing only objects having a particular characteristic into a container.
Systems according to the invention can help to accelerate and reduce the cost of pharmaceutical development. By rapidly sorting and depositing large numbers of live populations with particular characteristics, a sorting instrument according to the invention can allow many compounds to be tested on the sorted organisms in a given time period. By permitting particular types of multicellular organisms to be selected from large populations, individuals with infrequent mutations can be collected and studied more quickly. By permitting the selection and accurate deposition of large microspheres bearing test compounds the test organisms and test compounds can be rapidly and accurately combined. As a result, more experiments can be performed in the same amount of time, and these experiments can be performed at a lesser expense.